title: [Construction of shRNA expression vector targeting AATF and establishment of stably transfected U937 cells].
abstract: This study was aimed to construct the targeting AATF shRNA eukaryotic expression vector and establish the stably transfected U937 cell lines. The sequence of AATF mRNA was obtained from GenBank. After excluding homology, three plasmid expression vectors coding shRNA targeting 228 ∼ 249, 303 ∼ 324 and 443 ∼ 464 of AATF gene sequence were synthesized. Two terminals of shRNA carried BamHI and HindIII restriction sites. The selected nucleotides were cloned into the plasmid pSilencer 3.1-H1 neo respectively, and the resultant recombinant plasmids were named as pSA-1, pSA-2, pSA-3. The sequences of the recombinant plasmids were identified by DNA sequencing. The recombinant plasmids were transfected into the cell line U937 by electroporation with Neon(TM) Transfection System. The transfected cells were persistently screened under G418 (500 mg/L), and isolated with a limited dilution for 8 weeks. The inhibition of AATF mRNA and protein expression was respectively detected by RT-PCR and Western blot. The results indicated that RNAi eukaryotic expression vectors targeting AATF had correct reading frame and nucleotide sequence. Real-time PCR revealed that AATF shRNA effectively silenced mRNA expression of AATF. Western blot analysis found that AATF shRNA obviously suppressed protein expression of AATF (P < 0.05). It is concluded that the shRNA eukaryotic expression vector has been successfully constructed which can inhibit the expression of AATF, and the establishment of stably transfected U937 cell lines provide a original route for exploring the mechanism of AATF in human Leukemia further.
authors.affiliation: Department of Hematology, Xuzhou Medical College, Xuzhou, Jiangsu Province, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Lyu
authors.forename: Jiang
authors.initials: J
authors.lastname: Cao
authors.forename: Fan-Jing
authors.initials: FJ
authors.lastname: Meng
authors.forename: Ling-Yu
authors.initials: LY
authors.lastname: Zeng
authors.forename: Bin
authors.initials: B
authors.lastname: Pan
authors.forename: Chong
authors.initials: C
authors.lastname: Chen
authors.forename: Qing-Yun
authors.initials: QY
authors.lastname: Wu
authors.forename: Xu-Guang
authors.initials: XG
authors.lastname: Song
authors.forename: Zhen-Yu
authors.initials: ZY
authors.lastname: Li
authors.forename: Xiu-Ying
authors.initials: XY
authors.lastname: Pan
authors.forename: Kai-Lin
authors.initials: KL
authors.lastname: Xu
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D014162:Transfection; D020298:U937 Cells
chemical_list: C402637:AATF protein, human; D051017:Apoptosis Regulatory Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins